This proposed Phase II project will lead to a commercial product capable of transfecting individual target cells. This new biotool uses a patented microfabricated chip, called a nanofountain probe (NFP), to deliver molecules into live individual target cells by single-cell electroporation, which induces temporary nanopores in the cell membrane via the application of an electrical field that is localized at the tip of a microfludic cantilever on the NFP chip. This novel transfection technique is called nanofountain probe electroporation (NFP-E), and it has shown incredible promise for translation into a novel commercial biotool. Existing techniques for single-cell transfection, such as microinjection and electroporation by micropipette, require extensive operator training, are highly user-dependent and labor intensive, and routinely damage cells from excessive mechanical force on the cell membrane. The NFP cantilever reduces the mechanical stress on the cell membrane and can be tailored during fabrication. The long-term objective of this project is to develop the first sinle-cell transfection system that is easy to use, gentle on cells, and automated to offer relatively high throughput and eliminate user-dependent variability. The specific aims for this proposal are the following: (1) to optimize the design of product components for plug-and-play assembly and easy solution loading and recovery; (2) to develop software for image processing and analysis to simplify NFP-Electroporation by automating the process of locating target cells, detecting contact of the NPF tip with a cell membrane, and applying the electric field; and (3) to perform three key transfection experiments to demonstrate the potential of the NFP-E in R&D, to establish experimental protocols, collect statistics of efficiency/viability, verify specifications (efficiency, viability, throughput), and refine the image recognition software for a number of cell lines and primary cells. Accomplishing these aims will produce a product that could enable new capabilities for single-cell research and therapeutics including cell reprogramming/differentiation, cell-cell signaling, gene expression and protein interaction, cell-to-cell variability, drug discovery, personalized drug response diagnostics, and personalized medicine.